Biosynthetic binding proteins for immuno-targeting

ABSTRACT

Disclosed is a formulation for targeting an epitope on an antigen expressed in a mammal. The formulation comprises a pharmaceutically acceptable carrier together with a dimeric biosynthetic construct for binding at least one preselected antigen. The biosynthetic construct contains two polypeptide chains, each of which define single-chain Fv (sFv) binding proteins and have C-terminal tails that facilitate the crosslinking of two sFv polypeptides. The resulting dimeric constructs have a conformation permitting binding of a said preselected antigen by the binding site of each said polypeptide chain when administered to said mammal. The formulation has particular utility in in vivo imaging and drug targeting experiments.

The U.S. Government may have certain rights in the invention describedherein, by virtue of National Institutes of Health Grant No. UO1CA51880.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/133,804, filed Oct.7, 1993, U.S. Pat. No. 5,534,254 which is a continuation-in-partapplication U.S. Ser. No. 07/831,967, filed Feb. 6, 1992, abandoned, thedisclosure of which is incorporated herein by reference. Relatedapplications include: U.S. Ser. No. 08/356,786 filed Dec. 12, 1994 whichis a continuation of U.S. Ser. No. 07/831,967, filed Feb. 6, 1992, nowabandoned; U.S. Ser. No. 08/133,804, filed Oct. 7, 1993, now U.S. Pat.No. 5,534,254, which is a continuation-in-part of U.S. Ser. No.07/831,967, filed Feb. 6, 1992, now abandoned; U.S. Ser. No. 08/461,838filed Jun. 5, 1995, which is a divisional of U.S. Ser. No. 08/133,804,filed Oct. 7, 1993, which is a continuation-in-part of U.S. Ser. No.07/831,967, filed Feb. 6, 1992, now abandoned; and U.S. Ser. No.08/462,641, filed Jun. 5, 1995, which is a continuation of U.S. Ser. No.08/133,804, filed Oct. 7, 1993, which is a continuation-in-part of U.S.Ser. No. 07/831,967, filed Feb. 6, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates in general to novel biosynthetic compositions ofmatter having particular utility as in vivo targeting agents and morespecifically, to biosynthetic dimeric constructs of single-chain bindingproteins (sFv), conjugates thereof, and to methods for their production.

BACKGROUND OF THE INVENTION

The development of murine monoclonal antibodies and their proteolyticFab fragments has raised interest in their utility as diagnostic andtherapeutic reagents for in vivo imaging and drug targeting. However,successful in vivo targeting of radionuclides, drugs or toxins using 150kD intact antibodies or their 50 kD Fab fragments (an antibody fragmentconsisting of one light chain and approximately half of the heavy chainheld together by a single disulfide bond) have been restricted by thelimited penetration of these molecules from the vasculature into thetissues of interest, and by their slow clearance rates in vivo, whichfor IgG leads to behavior that requires several days to clear thebackground enough for imaging to be possible. Other disadvantages of theintact antibodies or their Fab fragments include: their immunogenicitywhen prepared from different species, their non-specific binding to manynormal tissues and organs, and the fact that they contain multipleproteolytic cleavage sites which result in their degradation duringtheir circulation in vivo.

Although Fv fragments, which consist of one V_(H) and one V_(L) domainheld together by noncovalent interactions, form the minimal region of anantibody that contains a complete antigen combining site, dissociationof the V_(H) and V_(L) domains in vivo can preclude their use astherapeutic or imaging agents. Although Moore et al., (U.S. Pat. No.4,642,334) and Glockshuber et al., (1990, Biochem. 29, 1362-1367)disclose attempts to stabilize these Fv fragments with engineeredintermolecular disulfide bonds, monovalent 50 kD Fab and Fab' fragmentshave, until recently, been the smallest proteins available for effectiveimmunotargeting.

Recently, single-chain Fv (sFv) polypeptide chains of about 27 kD havebeen developed containing covalently linked V_(H) -V_(L) polypeptides.The V_(H) - and V_(L) -domains are connected by a polypeptide linker.The resulting sFv polypeptide chains are also referred to in the art asbiosynthetic antibody binding sites or BABS and preferably are encodedby a single DNA sequence. For a detailed description of thesebiosynthetic polypeptide chains see for example, Huston et al., 1988,Proc. Nat. Aca. Sci. USA 85: 5879-5883 or U.S. Pat. Nos. 5,091,513 and5,132,405, all of which are hereby incorporated by reference. The sFvpolypeptide chains provide attractive alternatives to intactimmunoglobulins and Fab fragments due to their small size and theirstability at concentrations that typically promote dissociation ofnatural Fv fragments. U.S. Pat. Nos. 5,091,513 and 5,132,405; Huston etal., ((1991) Methods in Enzymology 203: 46-88; Huston et al (1993) Int.Rev. Immunol. 10: 195-217) disclose the utility of sFv polypeptides, aswell as single chain constructs synthesized from single DNA sequences,which may further comprise ancillary effector proteins, such as a secondsFv or a cytotoxic agent.

Pack et al. ((1992) Biochem 31: 1579-1584) disclose the construction of"mini-antibodies". The mini-antibodies are sFv polypeptide chains whichalso include an "oligomerization domain" at their C-termini, separatedfrom the sFv by a hinge region. The oligomerization domains compriseself-associating α-helices, for example, leucine zippers, that can befurther stabilized by additional disulfide bonds. The domains aredesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein.

PCT application PCT/US92/09965, published Jun. 10, 1993 also disclosesthe construction of bivalent sFv constructs, including crosslinkeddimers. However, the pharmacokinetic properties of these constructs orthose disclosed by Pack et al. are not measured in vivo.

PCT application PCT/US92/07986, published Apr. 1, 1993 disclosesbifunctional (Fab')₂ molecules composed of two Fab' monomers linkedthrough cysteine amino acids located at the C-terminus of the firstconstant domain of each heavy chain. PCT application PCT/US92/10140,published Jun. 10, 1993 also discloses bifunctional (Fab')₂ dimerswhich, in addition to the cysteine residues located in the hinge region,also contain C-terminal leucine zipper domains that further stabilizethe (Fab')₂ dimers. In both cases, the resulting (Fab')₂ dimers (≧100 kDin size), although smaller than intact immunoglobulins, aresignificantly larger than sFv polypeptides and are anticipated to haveslower tissue biodistribution and clearance rates following in vivoadministration.

Cumber et al. disclose the generation of (Fv-Cys)₂ heterodimers bychemically crosslinking two V_(H) -cys domains together (Cumber et al.,1992, J. Immunology 149B: 120-126). Although the crosslinked V_(H)chains appear to be stable, dissociation of the V_(L) polypeptides fromeach Fv reduces the pharmacological value of these constructs in vivo.

It is an object of the instant invention to provide biosyntheticconstructs having enhanced pharmacokinetic properties as in vivotargeting agents. In particular, it is an object of this invention toprovide biocompatible constructs having accelerated in vivobiodistribution and body clearance rates than that of antibodies orantibody fragments. It is another object of the invention to providebiosynthetic constructs having enhanced avidity in vivo, includingenhanced target tissue specificity and target tissue retention. Yetanother object is to provide dimeric biosynthetic constructs havingimproved tissue imaging and drug targeting properties in vivo. Stillanother object is to provide diagnostic and therapeutic formulationscomprising these constructs, having particular utility in the diagnosisand treatment of malignancies. Still another object is to provideconstructs having enhanced pharmacokinetic properties as in vivotargeting agents, particularly as in vivo imaging agents, for ovarianand breast tumor tissue.

These and other objects and features of the invention will be apparentfrom the description, figures and claims which follow.

SUMMARY OF THE INVENTION

In its broadest aspect, the invention features a formulation fortargeting an epitope on an antigen expressed in a mammal, where theformulation contains a pharmaceutically acceptable carrier incombination with a biosynthetic construct for binding at least onepreselected antigen. The dimeric construct has particular utility indiagnostic and therapeutic applications in vivo.

The invention features the synthesis and use of monomers and dimers ofpolypeptide constructs belonging to the class of proteins known assingle-chain Fv (sFv) polypeptides. The sFv proteins described hereinhave superior in vivo pharmacokinetic properties, including acceleratedtissue biodistribution and clearance rates relative to either intactIgG, (Fab)₂ dimers or Fab.

The dimeric biosynthetic construct of the invention contains two sFvpolypeptide chains defined herein as follows. Each sFv polypeptide chaincomprises an amino acid sequence defining at least two polypeptidedomains. These domains are connected by a polypeptide linker spanningthe distance between the C-terminus of one domain and the N-terminus ofthe other. The amino acid sequence of each domain includescomplementarity determining regions (CDRs) interposed between frameworkregions (FRs) where the CDRs and FRs of each polypeptide chain togetherdefine a binding site immunologically reactive with a preselectedantigen. Additionally, each biosynthetic binding site polypeptide chaincan have an amino acid sequence peptide bonded and thus contiguous withthe C-terminus of each polypeptide chain, referred to herein as a"C-terminal tail" sequence. The term "sFv'" refers hereinafter, to ansFv molecule containing such a C-terminal tail sequence. This tailsequence preferably does not contain an α-helical motif thatself-associates with another polypeptide chain of similar sequence butstill contains a means for covalently crosslinking two such polypeptidechains together. When the two sFv' polypeptide chains are crosslinkedtogether, the resulting dimeric construct has a conformation thatpermits the independent binding of a preselected antigen or antigens tothe binding site of each polypeptide chain in vitro and in vivo. Theresulting dimeric constructs have superior in vivo pharmacokineticproperties that include significantly enhanced avidity, includingenhanced target tissue retention and/or antigen localization properties,as compared with intact IgG, Fab, (Fab)₂ dimers or monomeric sFv.

As will be appreciated by those having ordinary skill in the art, thesequence referred to herein generally as a "C-terminal tail" sequence,peptide bonded to the C-terminus of an sFv and comprising means forcrosslinking two sFv polypeptide chains, alternatively may occur at theN-terminus of an sFv ("N-terminal tail") or may comprise part of thepolypeptide linker spanning the domains of an individual sFv. Thedimeric species created by the crosslinking of sFvs having thesealternative "tail" sequences also are contemplated to have aconformation permitting the in vivo binding of a preselected antigen bythe binding sites of each of the sFv polypeptide chains. Accordingly,descriptions of how to make and use sFv' monomers and dimers comprisinga C-terminal tail sequence are extended hereby to include sFv monomersand dimers wherein the tail sequence having crosslinking means occurs atthe N-terminus of an sFv or comprises part of the polypeptide linkersequence.

In one embodiment, both polypeptide chains bind the same epitope on apreselected antigen, and the resulting dimeric construct is termed a"homodimer." In another embodiment, the polypeptide chains binddifferent epitopes on a preselected antigen and the resulting dimericconstruct is termed a "heterodimer." In still another embodiment, thetwo polypeptide chains bind different epitopes on two different,preselected antigens.

The term "epitope", as used herein, refers to a portion of an antigenthat makes contact with a particular antibody or antibody analogue. In atypical protein, it is likely that any residue accessible from thesurface can form part of one or more antigenic determinants. The term"antigen", as used herein, refers to a molecule that can elicit animmune response and that can react specifically with correspondingantibodies or antibody analogues.

The term "domain", as used herein, refers to an amino acid sequence thatfolds into a single globular region in its native conformation, andwhich may exhibit discrete binding or functional properties. The term"polypeptide linker", as used herein, refers to an amino acid sequencethat links the C-terminus of one domain to the N-terminus of the otherdomain, while still permitting the two domains to maintain their properphysiologically active binding conformations. In a particular aspect ofthe invention, the currently preferred polypeptide linkers that connectthe C-terminus of one domain to the N-terminus of the other domaininclude part or all of amino acid sequence ((Gly)₄ Ser)₃ set forth inthe SEQ. ID. NO.: 7, or ((Ser)₄ Gly)₃ as set forth in SEQ. ID. NO.: 8.

The amino acid sequence of each of the polypeptide domains includescomplementarity determining regions interposed between frameworkregions. The term "complementarity determining regions" or "CDRs", asused herein, refer to amino acid sequences which together define thebinding affinity and specificity of the natural Fv region of a nativeimmunoglobulin binding site, or a synthetic polypeptide which mimicsthis function. CDRs are not necessarily wholly homologous tohypervariable regions of natural Fv molecules, and also may includespecific amino acids or amino acid sequences which flank thehypervariable region and have heretofore been considered framework notdirectly determinative of complementarity. The term "framework regions"or "FRs", as used herein, refers to amino acid sequences which are foundnaturally occurring between CDRs in immunoglobulins. These FR sequencesmay be derived in whole or part from the same immunoglobulin as theCDRs, or in whole or part from a different immunoglobulin. For example,in order to enhance biocompatibility of an sFv to be administered to ahuman, the FR sequences can be derived from a human immunoglobulin andso the resulting humanized sFv will be less immunogenic than a murinemonoclonal antibody.

The amino acid sequence of each variable domain includes three CDRsinterspersed between four FRs. The two polypeptide domains that definean sFv molecule contain CDRs interspersed between FRs which togetherform a binding site immunologically reactive with a preselected antigen.The term "immunologically reactive", as used herein, refers to thenoncovalent interactions of the type that occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. As used herein, the term "avidity" describes the stability ofa complex formed by a multivalent antibody or antibody analogue, withits binding conjugate. Also as used herein, the term "apparent avidity"describes the stability of a complex formed by an antibody or anantibody analogue with its binding conjugate as determined by in vivoimmunolocalization studies.

In a preferred aspect of the invention, the CDRs of the polypeptidechain can have an amino acid sequence substantially homologous with atleast a portion of the amino acid sequence of CDRs from a variableregion of an immunoglobulin molecule from a first species, together withFRs that are substantially homologous with at least a portion of theamino acid sequence of FRs from a variable region of an immunoglobulinmolecule from a second species. Preferably, the first species is mouseand the second species is human. The CDR sequences in the sFv'polypeptides are preferably substantially homologous to animmunoglobulin CDR retaining at least 70%, or more preferably 80% or90%, of the amino acid sequence of the immunoglobulin CDR, and alsoretains the immunological binding properties of the immunoglobulin.

Each sFv' molecule has a C-terminal polypeptide tail that has anon-self-associating structure and contains at least one crosslinkingmeans. Useful crosslinking means include derivatizable amino acid sidechains, particularly those selected from the group consisting ofcysteine, lysine, arginine, histidine, glutamate, aspartate, andderivatives and modified forms thereof. In a preferred aspect of theinvention, cysteine amino acids are incorporated into the C-terminaltail sequences as the crosslinking means. In another aspect of theinvention, the crosslinking means includes one or more amino acids thatcan be posttranslationally modified. For example, the crosslinking meanscan include one or more glycosylation sites, wherein the incorporatedcarbohydrate moieties can be crosslinked in vitro. Preferredglycosylation sequences include Asn-Xaa-Thr and Asn-Xaa-Ser, where Xaacan be any amino acid, wherein the carbohydrate is typically N-linked toasparagine or O-linked to serine or threonine.

Additionally, the tail also may comprise an amino acid sequence thatdefines a metal ion chelation motif, and which facilitates purificationof the sFv' monomers by metal ion affinity chromatography, such as theIMAC²⁺ chromatography system. Furthermore, chelation motifs can be usedfor binding detectable moieties, such as Technetium^(-99m) (^(99m) Tc)for in vivo imaging. Preferred examples of useful C-terminal tail aminoacid sequences wherein the crosslinking means is provided by thesulfhydryl group of a cysteine, include: Ser-Cys; (Gly)₄ -Cys; and(His)₆ -(Gly)₄ -Cys; set forth in the Sequence Listing as SEQ. ID. NOS.:9, 10 and 11, respectively. The (Gly)₄ -Cys sequence facilitates thecoordination of ^(99m) Tc by this tail.

In the present invention, monomeric sFv' molecules can be coupledtogether through the crosslinking means in the C-terminal tails to formeither homo- or heterodimeric (sFv')₂ species. The term "sFv coupler",as used herein, refers to the chemical bridge that links two sFv'polypeptide chains together to form a dimeric species. In a preferredaspect of the invention, where the crosslinking means is a cysteineresidue, the linkage is by a disulfide bond. Alternatively,sulfhydryl-specific homobifunctional crosslinking reagents, such asbismaleimidohexane, or heterobifunctional crosslinking reagents, can beused to join the two sFv' molecules together. sFv couplers ofpreselected length also can be designed to limit interaction between thetwo sFv' polypeptide chains or to optimize binding of two preselectedantigens, including, for example, multiple copies of a receptorexpressed on a cell surface in a mammal. An example of such a variablelength coupler includes the bismaleimidocaproyl amino acid (MCA)synthetic peptide bridge. Although, in a preferred aspect of theinvention a GlySer₃ Gly₂ Ser₃ Lys peptide spacer is used, in theory, anyamino acid sequence can be introduced into this type of chemical bridgewith a variety of reactive moieties at either end. Consequently, it ispossible to design specific linkage groups that can have a predeterminedlength and flexibility. If a substantially inflexible coupler isdesired, then for instance, a polylysine or polyproline peptide may beused. Another benefit of the MCA linkers over many other commerciallyavailable linkers is that they are soluble in water. Moreover, thechemical bridge also may be created to enhance the imaging ortherapeutic properties of the construct in vivo (vide infra). As will beappreciated by those having ordinary skill in the art, the separationdistance between, and interaction of, the sFv' monomers in a dimericconstruct of the invention also can be modulated by the judicious choiceof amino acids in the tail sequences themselves.

The dimeric constructs of this invention preferably target apharmacologically active drug (or other ancillary protein) to a site ofinterest utilizing the bivalent capability of the dimer. Examples ofpharmacologically active drugs include molecules that inhibit cellproliferation and cytotoxic agents that kill cells. The term "cytotoxicagent", as used herein, refers to any molecule that kills cells, andincludes anti-cancer therapeutic agents such as doxorubicin. Other,useful molecules include toxins, for instance, the toxic portion of thePseudomonas exotoxin, phytolaccin, ricin, ricin A chain, or diptheriatoxin, or other related proteins known as ricin A chain-like ribosomalinhibiting proteins, i.e., proteins capable of inhibiting proteinsynthesis at the level of the ribosome, such as pokeweed antiviralprotein, gelonin, and barley ribosomal protein inhibitor.

In such cases, one sFv' can be immunologically reactive with a bindingsite on an antigen at the site of interest, and the second sFv' in thedimer can be immunologically reactive with a binding site on the drug tobe targeted. Alternatively, the construct may bind one or more antigensat the the site of interest and the drug to be targeted is otherwiseassociated with the dimer, for example, crosslinked to the chemicalbridge itself. The biosynthetic dimeric constructs of this inventionalso may be used as part of human therapies to target cytotoxic cellssuch as cytotoxic T-lymphocytes, or pharmacologically active drugs to apreselected site. A bispecific (sFv')₂ heterodimer having specificityfor both a tumor antigen and a CD3 antigen, the latter of which ispresent on cytotoxic T-lymphocytes, thus could mediate antibodydependent cellular cytotoxicity (ADCC) or cytotoxic T-lymphocyte-inducedlysis of the tumor cells.

Still another bispecific dimeric construct having cytotoxic propertiesis a bispecific construct with one sFv' capable of targeting a tumorcell and the second sFv' having catalytic properties that binds aninactive drug, subsequently converting it into an active compound (seefor example, U.S. Pat. No. 5,219,732). Such a construct would be capableof inducing the formation of a toxic substance in situ. For example, acatalytic sFv' molecule having β-lactamase-like activity can be designedto bind and catalyze the conversion of an inactive lactam derivative ofdoxorubicin into its active form. Here the bispecific dimer, havingbinding affinities for both the preselected antigen and theinactive-lactam derivative, is administered to an individual and allowedto accumulate at the desired location. The inactive and nontoxiccytotoxin-lactam derivative then is administered to the individual.Interaction of the derivative with the bispecific (sFv')₂ heterodimer atthe site of interest releases the active form of the drug in situ,enhancing both the cytotoxicity and specificity of the drug.

The homo- and heterodimeric biosynthetic constructs also may include adetectable moiety bound either to the polypeptide chain, e.g., to thetail sequence, or to the chemical coupler. The term "detectable moiety",as used herein, refers to the moiety bound to or otherwise complexedwith the construct and which can be detected external to, and at adistance from, the site of the complex formation, to permit the imagingof cells or cell debris expressing a preselected antigen. Preferabledetectable moieties for imaging include radioactive atoms such asTechnetium^(-99m) (^(99m) Tc), a gamma emitter with a half-life of about6 hours. Non-radioactive moieties useful for in vivo magnetic resonanceimaging applications include nitroxide spin labels as well as lanthanideand transition metal ions which induce proton relaxation in situ. Inaddition to immunoimaging, the complexed radioactive moieties also maybe used in standard radioimmunotherapy protocols to destroy the targetedcell. Preferable nucleotides for high dose radioimmunotherapy includeradioactive atoms such as, ⁹⁰ Yttrium (⁹⁰ Yt), ¹³¹ Iodine (¹³¹ I) or ¹¹¹Indium (¹¹¹ In).

The sFv, sFv' and (sFv')₂ constructs disclosed herein have particularutility as in vivo targeting agents of tumor antigens, includingantigens characteristic of breast and ovarian malignancies, such as thec-erbB-2 or c-erbB-2 related antigens. Accordingly, these constructshave particular utility in diagnostic applications as imaging agents ofmalignant cells, and in therapeutic applications as targeting agents forcytotoxins and other cancer therapeutic agents. In one preferred aspectof the invention, the CDRs of the sFv or sFv' polypeptide chain have anamino acid sequence substantially homologous with the CDRs of thevariable region of any one of the following monoclonal antibodies:741F8, 520C9, and 454C11, all of which bind to c-erbB-2 orc-erbB-2-related antigens. Exemplary sFv' and sFv sequences having CDRscorresponding to the monoclonal antibodies 741F8 and 520C9 are set forthin the Sequence Listing SEQ. ID. NOS.: 1 and 5, respectively.

The term "c-erbB-2" refers to a protein antigen that is an approximately200 kD acidic glycoprotein having an isoelectric point of about 5.3 andhaving an extracellular domain overexpressed on the surface of tumorcells, such as breast and ovarian tumor cells in about 25% of cases ofbreast and ovarian cancer. A "c-erbB-2-related tumor antigen" is aprotein located on the surface of tumor cells, such as breast andovarian tumor cells and which is antigenically related to the c-erbB-2antigen. That is, the related antigen can be bound by an immunoglobulinthat is capable of binding the c-erbB-2 antigen (e.g. 741F8, 520C9, and454C11 antibodies. Related antigens also include antigens comprising anamino acid sequence that is at least 80% homologous, preferably 90%homologous, with the amino acid sequence of c-erbB-2 or an amino acidsequence encoded by a DNA that hybridizes under stringent conditionswith a nucleic acid sequence encoding c-erbB-2. As used herein,stringent hybridization conditions are those set forth in Sambrook, etal., 1989, Molecular Cloning; A Laboratory Manual 2nd ed. Cold SpringHarbor Press wherein the hybridization conditions, for example, include50% formamide, 5× Denhardt's Solution, 5×SSC, 0.1% SDS and 100 μg/mldenatured salmon sperm DNA and the washing conditions include 2×SSC,0.1% SDS at 37° C. followed by 1×SSC, 0.1% SDS at 68° C. An example of ac-erbB-2-related antigen is the receptor for the epidermal growthfactor.

In one embodiment, the biosynthetic antibody binding site is a humanizedhybrid molecule which includes CDRs from the mouse 741F8 antibodyinterposed between FRs derived from one or more human immunoglobulinmolecule. The CDRs that bind to the c-erbB-2 epitope can be found in theamino acid residue numbers 31-37, 52-68, 101-110, 159-169, 185-191 and224-233 in SEQ ID NOS.: 1 and 2. The hybrid molecule thus containsbinding sites which are highly specific for the c-erbB-2 antigen orc-erbB-2 related antigens held in proper immunochemical bindingconformation by human FR amino acid sequences, which are less likely tobe recognized as foreign by the human body.

The dimeric (sFv')₂ construct can either be homodimeric, wherein the CDRsequences on both monomers define the same binding site, orheterodimeric, wherein the CDR sequences of each sFv' monomer define adifferent binding site. An example of an (sFv')₂ heterodimer describedherein having specificity for both c-erbB-2 and digoxin epitopes can begenerated by combining the anti-c-erbB-2 sFv', shown in SEQ. ID. NOS.: 1and 2 with the anti-digoxin sFv', shown in SEQ. ID. NOS.: 3 and 4. TheCDRs that bind to the digoxin epitope can be derived from theanti-digoxin murine monoclonal antibody 26-10 (Huston et al., 1988,Proc. Natl. Acad. Sci. USA 85: 5879-5883) and can be found in the aminoacid residue numbers 32 through 36, 48 through 65, 101 through 107, 157through 170, 188 through 194 and 229 through 234 in the Sequence Listingas SEQ. ID. NOS.: 3 and 4.

Radioimaging or radioimmunotherapy of tumor tissues and malignant cellsare preferred aspects of this invention. Overexpression of tumorantigens such as c-erbB-2 and related cell surface antigens in malignantcells allows imaging of the malignant cell or tissue, whether it is welllocalized, has undergone metastasis or is exposed following cell lysis.The imaging method includes the steps of administering to a mammal aformulation comprising an sFv' or (sFv')₂ dimeric construct havingspecificity for the antigen tumor and containing a detectable moiety ata concentration sufficient to permit extracorporeal detection of theconstruct bound to the tumor antigen; and then detecting thebiosynthetic construct bound to the tumor antigen. The formulation canbe used to particular advantage in gamma scintigraphy or magneticresonance imaging. Overexpression of c-erbB-2 or related receptors onmalignant cells thus allows targeting of sFv' species to the tumorcells, whether the tumor is well-localized or metastatic. In addition,internalization of an sFv-toxin fusion protein permits specificdestruction of tumor cells bearing the overexpressed c-erbB-2 or relatedantigen.

The present invention discloses monomeric and dimeric biosyntheticconstructs having enhanced properties as in vivo targeting agents whencompared with intact monoclonal antibodies or their Fab fragments. Thedimeric biosynthetic constructs of the invention also permit the in vivotargeting of an epitope on an antigen with greater apparent avidity,including greater tumor specificity, tumor localization and tumorretention properties than that of the Fab fragment having the same CDRsas the construct. Furthermore, the dimeric constructs also permit the invivo targeting of an epitope on an antigen with a greater apparentavidity, including greater tumor localization and tumor retentionproperties, than either of the monomeric polypeptides individually.

The invention also includes methods for producing the homo- andheterodimeric biosynthetic constructs, which include the steps ofdesigning, constructing, expressing, purifying, and refolding themonomeric sFv' polypeptide chains in vitro, followed by joining twopolypeptide chains together through the crosslinking means in theC-terminal tail sequence, without relying on the tail structure tootherwise assist in dimer formation or enhance transport across amembrane. The invention also includes methods for imaging a preselectedantigen in a mammal expressing the preselected antigen. The antigen maybe expressed on a cell surface or may be released as part of the celldebris from a dying cell.

The foregoing and other objects, features and advantages of the presentinvention will be made more apparent from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself, will be more fully understoodfrom the following description, when read together with the accompanyingdrawings:

FIG. 1A is a schematic representation of a DNA construct encoding thesFv' biosynthetic binding protein of the invention;

FIG. 1B is a schematic representation of the polypeptide chain encodedby the DNA construct in FIG. 1A;

FIG. 2A is a schematic representation of a refolded sFv' protein in itsnative conformation;

FIG. 2B is a schematic representation showing two folded sFv'polypeptides covalently linked by a disulfide bond;

FIG. 3 is a graphic representation of an in vitro competition assaycomparing the c-erbB-2 binding activity of an Fab fragment of the 520C9monoclonal antibody (filled dots), with that of biosynthetic 520C9 sFvat two different stages of purification: mixture of folded and unfoldedsFv (+) or affinity-purified sFv (squares), and with a material that didnot bind to the affinity column (*);

FIG. 4 lists in tabular form the tumor:organ ratios calculated forvarious sF and sFv' species injected into tumor-containing mice;

FIG. 5 lists in tabular form the percentage of injected dose localizedto tumor tissue for various sFv and sFv's species; and

FIG. 6 is a graphic representation summarizing the comparative tumorretention properties of monomeric and dimeric forms of different sFv'constructs and Fabs represented by bars 1-6. The sFv' speciesrepresented by bars 1-5 are based on thr V regions of the 741F8monoclonal antibody. Bar 1 refers to intravenously (i.v.) administeredglutathionyl-(sFv'-SerCys) monomer, bar 2 to disulfide linked (sFv'-Gly₄-Cys)₂, bar 3 to MCA combined (sFv-Ser-Cys)₂, bar 4 to BMH cross-linked(sFv-Ser-Cys)₂, bar 5 to 741F8 Fab and bar 6 to the 26-10 disulfidelinked (sFv-Ser-Cys)₂.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that intravenously administered single-chain Fv(sFv) proteins exhibit superior in vivo pharmacokinetic propertiesrelative to intact monoclonal antibodies (IgG), (Fab)₂ dimers or Fabfragments. These pharmacokinetic properties include accelerated rates oftissue biodistribution, enhanced target tissue specificity, andexceptionally fast clearance rates. The sFv constructs can be designedto bind to preselected antigens and to have particular utility for invivo immunoimaging and immunotherapy applications. In addition, it alsohas been discovered that dimeric forms of the constructs, which do notrely on self-associating tail sequences for dimerization or transportacross a membrane, can be easily prepared and have improved targettissue localization properties, target tissue retention propertiesand/or avidity for their targets in vivo, relative to monomeric sFv',Fab fragments or intact IgG.

In its broadest aspect, the invention features a formulation fortargeting an epitope on an antigen expressed in a mammal. Theformulation contains a pharmaceutically acceptable carrier incombination with a dimeric biosynthetic construct for binding at leastone preselected antigen. The preselected antigen either may be anantigen expressed on the surface of a cell or an intracellular componentexposed upon lysis of the cell. The sFv, sFv' and (sFv')₂ constructsdisclosed herein have particular utility as in vivo targeting agents fordetecting malignant cells in a mammal. In a particularly usefulembodiment, the constructs disclosed can be used to target the c-erbB-2or c-erbB-2-related antigens which are overexpressed in certain breastand ovarian cancers. In another embodiment, radioimmunotargeting usingradiolabeled (sFv')₂ constructs will be useful for therapeutic as wellas diagnostic applications.

Provided below are detailed descriptions of biosynthetic sFv, sFv' and(sFv')₂ dimers, useful in the compositions and methods of the invention,together with methods for their construction and administration. Alsoprovided are numerous, non-limiting examples which demonstrate thesuitability of these constructs as in vivo targeting reagents fordiagnostic and therapeutic applications. More specifically, the examplesdemonstrate: the construction and expression of sFv polypeptides(Example 1); the renaturation, dimerization and purification of sFv'proteins (Example 2); and the immunoreactivity of the monomeric anddimeric sFv proteins (Example 3).

Construction of Biosynthetic Single-chain Fv Proteins.

Each of the sFv and sFv' proteins have amino acid sequences that defineat least two polypeptide domains. The polypeptide domains are connectedby a polypeptide linker spanning the distance between the C-terminus ofone domain and the N-terminus of the other. The amino acid sequence ofeach domain includes complementarity determining regions (CDRs)interposed between framework regions (FRs), where the CDRs and FRs ofeach polypeptide chain together define a binding site immunologicallyreactive with a preselected antigen.

In the case of the sFv' proteins, each polypeptide chain has anadditional C-terminal tail amino acid sequence having a substantiallynon-self-associating structure. More specifically, this is a sequencethat does not interact appreciably with a similar sequence underphysiological conditions, as is the case for example with the α-helicalleucine zipper motifs found in DNA binding proteins. Each tail sequencealso contains a means for crosslinking two such sFv' polypeptide chainstogether to form an (sFv')₂ dimer. The resulting (sFv')₂ dimers haveconformations which permit the in vivo binding of the preselectedantigen by the binding sites of each of the polypeptide chains.

The sFv' constructs of this invention can be further understood byreferring to the accompanying FIGS. 1 and 2. FIG. 1A is a schematicrepresentation of the DNA construct, and FIG. 1B is a schematicrepresentation of the resulting encoded polypeptide chain. FIG. 2 is aschematic representation of the folded sFv' monomer (FIG. 2A) and thedimeric (sFv')₂ construct (FIG. 2B). A single-chain Fv (sFv')polypeptide, shown in FIGS. 1 and 2A, comprises: a heavy chain variableregion (V_(H)) 10, and a light chain variable region, (V_(L)) 14,wherein the V_(H) and V_(L) domains are attached by polypeptide linker12. The binding domains defined by V_(L) and V_(H) include the CDRs 2,4, 6 and 2', 4', 6', respectively, and FRs 32, 34, 36, 38 and 32', 34',36', 38', respectively which, as shown in FIG. 2, together define animmunologically reactive binding site or antigenic determinant, 8.Furthermore, the CDRs and FRs may be derived from differentimmunoglobulins (see infra). The sFv' molecules also contain aC-terminal tail amino acid sequence, 16, comprising an amino acidsequence that will not self-associate with a polypeptide chain having asimilar amino acid sequence under physiological conditions, and whichcontains a means, 18, for the site-directed crosslinking of two suchtail sequences. In a currently preferred embodiment, represented inFIGS. 1 and 2, the crosslinking means is the sulfhydryl group of acysteine amino acid. In the monomeric form of the sFv' the crosslinkingmeans, 18, may be blocked by a blocking group, 20. For instance, theblocking group may be a glutathionyl moiety when the crosslinking means,18, is a cysteine amino acid.

As will be appreciated by those having ordinary skill in the art, thesequence referred to herein generally as a "C-terminal tail" sequence,peptide bonded to the C-terminus of an sFv and comprising means forcrosslinking two sFv polypeptide chains, alternatively may occur at theN-terminus of an sFv ("N-terminal tail") or may comprise part of thepolypeptide linker spanning the domains of an individual sFv. Thedimeric species created by the crosslinking of sFvs having thesealternative "tail" sequences also are contemplated to have aconformation permitting the in vivo binding of a preselected antigen bythe binding sites of each of the sFv polypeptide chains. Accordingly,descriptions of how to make and use sFv' monomers and diners comprisinga C-terminal tail sequence are extended hereby to include sFv monomersand dimers wherein the tail sequence having crosslinking means occurs atthe N-terminus of an sFv or comprises part of the polypeptide linkersequence.

The CDR and FR polypeptide segments are designed empirically based onsequence analysis of Fv regions of preexisting antibodies, such as thosedescribed in U.S. Pat. No. 4,753,894, hereby incorporated by reference.Numerous examples of sFv polypeptide chains now exist in the art and aresummarized in Huston et al., 1993, Intern. Rev. Immunol. 10: 195-217,hereby incorporated by reference.

The sFv and sFv' polypeptide chains of the invention are biosynthetic inthe sense that they are synthesized, transfected into a cellular host,and protein expressed from a nucleic acid containing genetic sequencesbased in part on synthetic DNA. Synthetic DNA is understood to includerecombinant DNA made by ligation of fragments of DNA derived from thegenome of a hybridoma, mature B cell clones, a cDNA library derived fromnatural sources, or by ligation of plural, chemically synthesizedoligonucleotides. The proteins of the invention are properlycharacterized as "antibody binding sites", in that these syntheticsingle polypeptide chains are able to refold into a 3-dimensionalconformation with specificity and affinity for a preselected epitope onan antigen.

A detailed description for engineering and producing sFv proteins byrecombinant means appears in U.S. Pat. No. 5,091,513 claiming priorityfrom U.S. Ser. No. 052,800, filed May 21, 1987, assigned to CreativeBioMolecules, Inc., hereby incorporated by reference. The polypeptidechains of the invention are antibody-like in that their structure ispatterned after regions of native antibodies known to be responsible forantigen recognition.

The single-chair polypeptide chains of the invention are first derivedat the DNA level. The sFv DNAs are preferably expressed in E. coli, theresulting polypeptide chains being solubilized from inclusion bodies,refolded in vitro, labeled with a detectable moiety, such as ^(99m) Tc,and dimerized to form a biosynthetic (sFv')₂ construct. Of course, theconstructs disclosed herein may also be engineered for secretion fromthe host cell, for example, secretion into the periplasmic space of anE. coli cell, as described by Pack and Pluckthun, (Biochem., 1992, 31:1579-1584), or into the culture supernatant of a mammalian cell (forexample, as described by Traunecker, et al., 1991, EMBO J. 10:3655-3659).

The ability to design the single polypeptide chains of the inventiondepends on the ability to identify Fv binding domains of interest, andto obtain the DNA encoding these variable regions. Hybridoma technologyenables the production of cell lines that secrete antibodies toessentially any desired substance that elicits an immune response. Forexample, U.S. Pat. No. 4,753,894 describes some monoclonal antibodies ofinterest which recognize c-erbB-2 related antigens on breast cancercells, and explains how such antibodies were obtained. One monoclonalantibody that is particularly useful in targeting the c-erbB-2 antigenis 741F8 (Bjorn et al., 1985, Cancer Res. 45: 1214-1221; U.S. Pat.4,753,894). This antibody specifically recognizes the c-erbB-2 antigenexpressed on the surface of various tumor cell lines, and exhibits verylittle binding to normal tissues. Other monoclonal antibodies that bindc-erbB-2 or related antigens include 520C9 and 454C11 (Frankel et al.,1985, J. Biol. Resp. Modif. 4: 273-286; Ring et al., 1989, Cancer Res.49: 3070-3080, Ring et al., 1991, Molec. Immunol. 28: 915-917; U.S. Pat.No. 4,753,894 and 5,169,774). sFv' sequences with the desiredspecificity can also be derived from phage antibody cloning ofcombinatorial V gene libraries. Such sequences could be based on cDNAderived from mice preimmunized with tumor cell membranes bearingc-erbB-2 or related antigenic fragments, (See, for example, Clackson etal, (1991) Nature 352: 624-628).

The process of designing DNA encoding the single polypeptide chain ofinterest can be accomplished as follows. Either synthetic DNA duplexescan be ligated together to form a synthetic gene or relevant DNAfragments can be cloned from libraries. In the latter procedure, mRNAencoding the light and heavy chains of the desired immunoglobulin may beisolated from hybridomas producing the immunoglobulin and reversetranscribed into cDNA. The V_(H) and V_(L) genes subsequently can beisolated by standard procedures, for instance, by colony hybridizationof cDNA libraries (see for example, Sambrook et al., eds., 1989,Molecular Cloning, Cold Spring Harbor Laboratories Press, N.Y.) or bypolymerase chain reaction (PCR) (see for example, Innis et al., eds.,1990, PCR Protocols, A guide to methods and applications, AcademicPress). Both procedures are well known in the art.

Still another approach involves the design and construction of syntheticvariable domain genes encoding a predetermined, specific Fv bindingsite. For example, with the help of a computer program, such asCompugene, one may design and directly synthesize native or near-nativeFR sequences from a first antibody molecule, and CDR sequences from asecond antibody molecule. The resulting V_(H) and V_(L) gene sequencescan then be genetically linked together by means of a linker connectingthe C-terminus of one chain with the N-terminus of the other.

Practice of the invention enables the design and synthesis of varioussingle-chain binding proteins, all of which are characterized by aregion having affinity for a preselected epitope on an antigen. Otherregions of the biosynthetic protein are designed with the particularplanned utility of the protein in mind. Thus, if the reagent is designedfor intravascular use in mammals, the FRs may include amino acidsequences which are similar or identical to at least a portion of the FRamino acid sequences of antibodies native to that species. The aminoacid sequences constituting the CDRs may be analogous to the sequencesfrom a second, different preexisting antibody having specificity for theantigen of interest (e.g. a murine or other human IgG). Alternatively,the CDRs and FRs may be copied in their entirety from a singlepre-existing monoclonal antibody cell line or a desirable sFv speciesmay be cloned from a repertoire library derived from preimmunized ornaive animals.

It is noted however, that the linear arrangement of the V_(L) and V_(H)domains in the DNA sequence of FIG. 1 is not critical. That is, althoughthe sequence represented in FIG. 1A encodes a heavy chain variableregion followed by the light chain variable region, as will beappreciated by those skilled in the art, the sFv may be constructed sothat the light and heavy chain domains are in reverse order.

As mentioned above, the V_(H) and V_(L) domains of the sFv are linked inthe gene construct by means of a linker 12 (FIG. 1A). The linker shouldbe at least long enough (e.g., about 10 to 15 amino acids or at least 40Angstroms in length) to permit domains 10 and 14 to assume their properconformations and interdomain relationships. The linkers preferablycomprise hydrophilic amino acids that assume an unstructuredconfiguration under physiological conditions, and are free of residueshaving large side groups that could interfere with proper folding of theV_(H), V_(L), or pendant chains. Examples of currently preferred linkersinclude either part or all of the amino acid sequences ((Gly)₄ Ser)₃ and((Ser)₄ Gly)₃, set forth in the Sequence Listing as SEQ. ID. NOS.: 7 and8, respectively. The linker may also include an amino acid sequencehomologous to a sequence identified as "self" by the species into whichit will be introduced, particularly if a therapeutic application isintended.

Considerations for Suitable C-terminal Tail Sequences.

As mentioned above, the sFv' polypeptide chains further comprise aC-terminal tail containing at least one amino acid that can bederivatized or posttranslationally modified to enable crosslinking oftwo such sFv' monomers. In preferred aspects of the invention, the tailsequences include one or more of the sequences Ser-Cys, (Gly)₄ -Cys and(His)₆ -(Gly)₄ -Cys, set forth in the Sequence Listing as SEQ. ID. NOS.:9, 10, and 11, respectively. The C-terminal tails preferably do not formα-helical structures which self-associate under physiologicalconditions, such as the α-helical leucine zipper motifs found in DNAbinding proteins (O'Shea et al., 1989, Science 243: 538-542, O'Shea etal., 1991, Science 254: 539-544) or the four-helix bundle motifs foundin recombinant ion channels (Hill et al., 1990, Science 294: 543-546).

Suitable derivatizable amino acid side chains may be selected from thegroup consisting of cysteine, lysine, arginine, histidine, glutamate,aspartate and derivatives or modified forms thereof. In a preferredaspect of the invention, cysteine amino acids are incorporated into theC-terminal tail sequences as the crosslinking means.

Also envisioned to be useful are posttranslationally modified aminoacids that can be crosslinked in vitro. More specifically, the glycosylmoieties present on glycosylated amino acids, following secretion out ofthe cell, can be covalently attached in vitro using bifuntional linkerson standard sugar chemistry (see for example, E. A. Davidson (1967)Carbohydrate Chemistry, Holt, Kinehart and Winston, N.Y.; W. J. Lennarz(1980) The Biochemsitry of Glycoproteins and Proteoglycans, PlenumPress, N.Y.). Particularly useful glycosylation sites include thesequences Asn-Xaa-Thr and Asn-Xaa-Ser, wherein Xaa is any amino acid.Where crosslinking of glycosyl moieties is contemplated, theglycosylation sequences need not include a cysteine.

The tail also may comprise an amino acid sequence defining an ionchelation motif which can be used as part of a purification protocol forisolating of the sFv' monomers by metal ion affinity chromatography(e.g., by means of a (His)₆ tail on an IMAC chromatography column), aswell as for chelating ions of detectable moieties such asTechnetium^(-99m) or ¹¹¹ Indium for in vivo imaging applications.

sFv' Coupler Considerations.

In the present invention, two monomeric sFv' proteins are crosslinkedtogether through their C-terminal tails to form an (sFv')₂ dimer. Theterm "sFv coupler", as used herein, refers to chemical bridges that jointhe crosslinking residues in each of the sFv' molecules.

In one preferred aspect of the invention, where the crosslinking residueis a cysteine residue, the chemical bridge can be a disulfide bond.Alternatively, sulfhydryl-specific crosslinking reagents can be used tojoin two sFv' molecules together. An example of such a cysteine-specificchemical bridge includes the bifunctional crosslinking reagentbismaleimidohexane (BMH), a water insoluble linker that can be obtainedfrom Pierce, Rockford, Ill. Other bifunctional crosslinking agentsinclude heterobifunctional crosslinkers which can be used to join twosFv' species together where the crosslinking residues in each of thesFv' C-terminal tail sequences are different, such as, a C-terminalcysteine on one sFv' and a C-terminal lysine on the other. Usefulheterobifunctional crosslinking agents include4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-toluene (SMPT) orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), both of which canbe obtained from Pierce, Rockland, Ill.

sFv couplers of variable length also can be prepared to limit stericinteraction of two coupled sFv' proteins. An example of such an sFvcoupler includes a peptide bridge, such as the water solublebismaleimidocaproyl amino acid (MCA) linker. Although in a preferredaspect of the invention, an MCA-GlySer₃ Gly₂ Ser₃ Lys-MCA linker isused, in theory, any amino acid sequence can be introduced into thistype of chemical bridge-spacer group.

Suitable MCA-peptide chemical bridges can be synthesized on polystyreneresins functionalized with hydroxymethylphenoxyacetic acid (HMP) toallow formation of free acids at the C-terminus following deblocking.During the synthesis of the preferred peptide sequence Gly-Ser₃ -Gly₂-Ser₃ -Lys the C-terminal lysine is esterified to the resin and otheramino acids are added as N-α-Fmoc protected derivatives.DIC/hydroxybenzotriazol activated amino acids are coupled for 90 minutesafter which the N-α-Fmoc protected groups are deprotected with 20%piperidine in dimethylformamide (DMF). Upon completion of the synthesis,the peptide is cleaved from the resin and deprotected with 95%trifluoroacetic acid (TFA) in water. The crude peptide then is dissolvedin 0.1M phosphate buffer pH 7 and reacted overnight at room temperaturewith maleimidocaproic acid N-hydroxysuccinimide ester. The resultinghomobifunctional peptide crosslinker can be purified by reverse-phaseHPLC, for example, on a Vydac 1×25 cm column usingacetonitrile/water/TFA buffers.

With this procedure, it is possible to generate linkers having specificlengths and flexibilities. Since polypeptides having particularsecondary structures and flexibilities are well documented in the art,it is possible to judiciously design the peptide couplers with optimallength and flexibility to optimize binding to two preselected antigenson a cell surface. As will be appreciated by those skilled in the art,the separation distance between, and interaction of, the sFv' monomersin a dimeric construct of the invention also can be modulated by thejudicious choice of amino acids in the tail sequences themselves.

Dimer Considerations.

Using the approaches described above, (sFv')₂ dimers readily can beprepared wherein the resulting dimers either can be homodimeric, wherethe CDR sequences define the same epitope binding site, orheterodimeric, where the CDR sequences of each sFv' monomer definedifferent epitope binding sites.

The dimeric constructs of this invention preferably target apharmacologically active drug (or other ancillary protein) to a site ofinterest utilizing the bivalent capability of the dimer. Examples ofpharmacolcogically active drugs include molecules that inhibit cellproliferation and cytotoxic agents that kill cells. Other, usefulmolecules include toxins, for instance, the toxic portion of thePseudomonas exotoxin, phytolaccin, ricin, ricin A chain, or diptheriatoxin, or other related proteins known as ricin A chain-like ribosomalinhibiting proteins, i.e., proteins capable of inhibiting proteinsynthesis at the level of the ribosome, such as pokeweed antiviralprotein, gelonin, and barley ribosomal protein inhibitor.

In such cases, one sFv' can be immunologically reactive with a bindingsite on an antigen at the site of interest, and the second sFv' in thedimer can be immunologically reactive with a binding site on the drug tobe targeted. For example, the (sFv')₂ dimers may have specificity forboth c-erbB-2 and a pharmacologically active drug or cytotoxic agent.The resulting dimer can thus target the agent or drug to tissuesexpressing the c-erbB-2 antigen in vivo. Alternatively, the constructmay bind one or more antigens at the the site of interest and the drugto be targeted is otherwise associated with the dimer, for example, bycrosslinking to the chemical bridge itself.

Other bispecific (sFv')₂ constructs having particular utility intargeting malignant cells, include constructs wherein one hasspecificity for a c-erbB-2 or related tumor antigen, and the seconddeterminant has specificity for a different cell surface protein, suchas the CD3 antigen found on cytotoxic T-lymphocytes. The heterodimeric(sFv')₂ construct then could mediate antibody dependent cellularcytotoxicity (ADCC) or cytotoxic T-lymphocyte-induced lysis of the tumorcells expressing the c-erbB-2 antigen.

Still another bispecific dimeric construct having cytotoxic propertiesis a bispecific construct with one sFv' capable of targeting a tumorcell and the second being a catalytic sFv' that binds an inactive drug,and subsequently converts it into an active compound (see for example,U.S. Pat. No. 5,219,732). Such a construct would be capable of inducingthe formation of a toxic substance in situ. For example, a catalyticsFv' molecule having β-lactamase-like activity can be designed to bindand catalyze the conversion of an inactive lactam derivative ofdoxorubicin into the active, cytotoxic form. Here the bispecific dimer,having binding affinities for both the preselected antigen and thecytotoxic-lactam derivative, is administered to an individual andallowed to accumulate at the desired location. The inactive, nontoxiccytotoxin-lactam derivative then is administered to the individual. Whenthe derivative is complexed with the bispecific (sFv')₂ heterodimer insitu the active form of the drug is released, enhancing both thecytotoxicity and specificity of the drug.

Hybrid sFv' Considerations.

In a preferred aspect of the invention a humanized single-chain Fv isenvisioned whereby the recombinant sFv' contains CDRs of the murine741F8 antibody interposed between human FR sequences to generate ahumanized c-erbB-2 binding protein. The humanized Fv would be capable ofbinding c-erbB-2 while eliciting little or no immune response whenadministered to a patient. A nucleic acid sequence encoding a humanizedsFv may be designed and constructed as follows.

FR regions identified by homology searches of the GenBank database canbe introduced into an sFv of interest by site-directed mutagenesis toreproduce the corresponding human sequence. Alternatively, homologoushuman V_(H) and V_(L) sequences can be derived from a collection ofPCR-cloned human V regions, after which the human FR sequences can beligated with murine CDR regions to create humanized V_(L) and V_(H)genes. A humanized sFv hybrid thus can be created, for instance, wherethe human FR regions of the human myeloma antibody are introducedbetween the murine CDR sequences of the murine monoclonal antibody741F8. The resulting sFv, containing the sequencesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, contains a murine binding site in ahuman framework.

By directly sequencing the DNA or RNA in a hybridoma secreting anantibody to a preselected antigen, or by obtaining the sequence from theliterature, one skilled in the art can essentially produce any desiredCDR and FR sequence. Expressed sequences subsequently may be tested forbinding and empirically refined by exchanging selected amino acids inrelatively conserved regions, based on observations of trends of aminoacid sequences in data bases and/or by using computer-assisted modelingtechniques. Significant flexibility in V_(H) and V_(L) design ispossible because alterations in amino acid sequences may be made at theDNA level.

Of course, the processes for manipulating, amplifying, and recombiningDNAs that encode amino acid sequences of interest are generally wellknown in the art (see, for example, Sambrook et al., 1989, MolecularCloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Press), andtherefore, are not described in detail herein. Similarly, methods foridentifying the isolated V genes encoding antibody Fv regions ofinterest are well understood and are described in the patent and otherliterature.

Expression of Recombinant sFv Proteins.

The resulting sFv DNA constructs then are integrated into expressionvectors and transfected into appropriate host cells for proteinexpression. After being translated, the protein may be purified from thecells themselves or recovered from the culture medium.

The expression vectors also may include various sequences to promotecorrect expression of the recombinant protein. Typical sequences includetranscription promoters and termination sequences, enhancer sequences,preferred ribosome binding site sequences, preferred mRNA leadersequences, preferred protein processing sequences, preferred signalsequences for protein secretion, and the like. The DNA sequence encodingthe gene of interest also may be manipulated to remove potentiallyinhibiting sequences or to minimize unwanted secondary structureformation. The resulting synthetic genes can be expressed in appropriateprokaryotic hosts such as various strains of E. coli, or in eucaryotichosts such as Chinese hamster ovary cells (CHO), mouse myeloma,hybridoma, transfectoma, and human myeloma cells. The currentlypreferred expression system for the present invention is E. coli, asdisclosed herein.

When the gene is to be expressed in E. coli, it is cloned into anexpression vector downstream of a strong promoter sequence, such as Trpor Tac, and optionally also may include a gene coding for a leaderpolypeptide, such as the fragment B (FB) of staphylococcal protein A.The resulting fusion protein, when expressed, accumulates in retractilebodies (also known as inclusion bodies) in the cytoplasm, and may beharvested after disruption of the cells by French press or sonication.The proteins then are solubilized, and refolded in vitro, as describedherein. Where the construct is engineered as a fusion protein, theprotein is solubilized and the leader sequence preferably cleaved beforerenaturation. The cleavage site for the leader sequence preferably isimmediately adjacent to the sFv polypeptide chain and includes one aminoacid or a sequence of amino acids exclusive of any one amino acid oramino acid sequence found in the amino acid structure of the singlepolypeptide chain.

The cleavage site preferably is designed for specific cleavage by aselected agent. Endopeptidases are preferred, although non-enzymatic(e.g., chemical) cleavage agents may be used. Many useful cleavageagents, for instance, cyanogen bromide (CNBr), dilute acid, trypsin,Staphylococcus aureus V-8 protease, post-proline cleaving enzyme, bloodcoagulation Factor Xa, enterokinase, and renin, recognize andpreferentially or exclusively cleave at particular cleavage sites. Onecurrently preferred peptide sequence cleavage agent is V-8 protease. Thecurrently preferred cleavage site is at a Glu residue. Other usefulenzymes recognize multiple residues as a cleavage site, e.g., factor Xa(Ile-Glu-Gly-Arg) or enterokinase (Asp-Asp-Asp-Asp-Lys). Dilute acidpreferentially cleaves the peptide bond between Asp-Pro residues, andCNBr in acid cleaves after Met, unless it is followed by Tyr.

Alternatively, the engineered gene may be incorporated into a vectorwithout a sequence encoding a leader polypeptide, and the engineeredgene expressed to produce a polypeptide chain that is secreted into theE. coli periplasmic space. The secreted protein then can be isolatedand, optionally, purified further using standard methodologies. (See,for example, Pack et al. (1992) Biochem 31:1579-1584.)

If the engineered gene is to be expressed in eucaryotic hybridoma cells,the conventional expression host for immunoglobulins, the genepreferably is inserted into an expression vector containing, forexample, the immunoglobulin promoter, a secretion signal, andimmunoglobulin enhancers. This plasmid also may contain sequencesencoding other polypeptide chains, including part or all of a toxin,enzyme, cytokine, or hormone. The gene then is transfected into myelomacells via established electroporation or protoplast fusion Methods. Thetransfected cells then may express V_(H) -linker-V_(L) -tail or V_(L)-linker-V_(H) -tail single-chain Fv' polypeptide chains.

The sFv polypeptide chains can be expressed as either inactive or activepolypeptide chains. Spontaneously refolded sFv polypeptide chains can beobtained from either prokaryotic or eukaryotic expression systems whenthe polypeptide chains are secreted for instance, either into the E.coli periplasmic space or the mammalian cell culture medium. Thesespontaneously refolded polypeptide chains readily can be purified byaffinity chromatography. Where the sFv polypeptide chains are obtainedin an unfolded, inactive sFv form, for instance, when overexpression ofthe sFv polypeptide chain in E. coli results in the formation ofinclusion bodies, the proteins can be refolded in vitro. Briefly,inclusion bodies are harvested by centrifugation, the sFv, solubilizedwith denaturants such as guanidine hydrochloride (GuHCl) or urea, andthen refolded by dilution of the denaturant under appropriate redox(reduction/oxidation) conditions (see below). The refolded sFvpolypeptide chains then can be purified by affinity chromatography.Details for the isolation of inclusion bodies, solubilization andrenaturation of the sFv polypeptide chains are well known in the art(see for example, U.S. Pat. No. 5,091,513 and Huston et al., 1988,supra).

Dimerization and Purification of the sFv Polypeptides

The sFv' monomers of the present invention can be dimerized in vivo orin vitro. In the in vivo approach, two sFv' genes can be cotransfectedinto the host cell wherein the coexpressed sFv' polypeptide chainsspontaneously dimerize. Alternatively, the refolded, secreted sFv'polypeptide chain monomers can be isolated from two expression hosts andsubsequently dimerized in vitro.

In a preferred aspect of the invention, the sFv' polypeptide chainscomprising a single cysteine C-terminal tail residue are expressed in E.coli and form inclusion bodies. The resulting sFv' polypeptide chainsare solubilized with denaturants and renatured in vitro, either in thepresence or absence of exogenously added glutathione. Surprisingly, theadditional C-terminal cysteine residues apparently do not interfere withthe refolding process. In some cases however, sFv and sFv' constructsmay refold poorly in vitro. These constructs can be "preoxidized prior"to refolding as taught in Huston et al., (1991) Meth. Enzymol.203:46-88, or, alternatively, the polypeptide chains can be secretedacross a membrane bilayer. The latter process spontaneously separatesthe extra C-terminal cysteine residue from the cysteine residuesnormally found in the Fv domain, minimizing inappropriate disulfide bondformation. Secretion is the preferred method if the sFv' constructsrefold poorly in vitro.

Following renaturation of the sFv' monomers, (sFv')₂ dimers readily canbe prepared in vitro by air oxidation if cysteine amino acids arepresent in the C-terminal tail sequences. Alternatively, sulfhydrylspecific crosslinking reagents, for instance, the BMH crosslinker or theMCA-peptide-MCA bridge may be used to covalently couple two sFv' chains.The resultant homo or heterodimers, then can be purified by standardsize exclusion chromatography. However, when (sFv)₂ heterodimers arerequired, then a preferred purification protocol uses a sequential twostep affinity chromatography procedure. Briefly, the heterodimer isexposed to a first chromatographic system having an epitope thatinteracts specifically with one sFv of the heterodimer. The eluantcontaining the heterodimer is then exposed to a second system having anepitope that interacts specifically with the other sFv. For specificdetails of the dimerization and purification procedures, see Example 2.

Considerations for In Vivo Administration.

The dimeric constructs may be administered either by intravenous orintramuscular injection. Effective dosages for the single-chain Fvconstructs in antitumor therapies or in effective tumor imaging can bedetermined by routine experimentation, keeping in mind the objective ofthe treatment.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions. In all cases, the form must be sterile andmust be fluid so as to be easily administered by syringe. It must bestable under the conditions of manufacture and storage, and must bepreserved against the contaminating action of microorganisms. This may,for example, be achieved by filtration through a sterile 0.22 micronfilter and/or lyophilization followed by sterilization with a gamma raysource.

Sterile injectable solutions are prepared by incorporating the desirableamount of the constructs, disclosed herein, into an appropriate solvent,such as sodium phosphate-buffered saline (PBS), followed by filtersterilization. As used herein, "a physiologically acceptable carrier"includes any and all solvents, dispersion media, antibacterial andantifungal agents that are non-toxic to humans, and the like. The use ofsuch media and agents as pharmaceutically active substances are wellknown in the art. The media or agent must be compatible with maintenanceof proper conformation of the single-chain Fv polypeptide chains, andits use in the therapeutic compositions. Supplementary activeingredients can also be incorporated into the compositions.

A preferred remotely detectable moiety for in vivo imaging includes theradioactive atom Technetium^(-99m) (^(99m) Tc), a gamma emitter with ahalf-life of about 6 hours. Non-radioactive moieties also useful inimaging include nitroxide spin labels as well as lanthanide andtransition metal ions all of which induce proton relaxation in situ. Inaddition to immunoimaging, the complexed radioactive moieties may beused in standard radioimmunotherapy protocols to destroy the targetedcell. Preferred nucleotides for high dose radioimmunotherapy include theradioactive atoms ⁹⁰ Yttrium (⁹⁰ Yt), ¹³¹ Iodine (¹³¹ I) and ¹¹¹ Indium(¹¹¹ In).

Either the single polypeptide chain sFv' itself, or the spacer groupsfor linking the sFv' constructs can be labeled with radioisotopes suchas ¹³¹ I, ¹¹¹ In and ^(99m) Tc. ^(99m) Tc and ¹¹¹ In are preferredbecause they can be detected with gamma cameras and have favorablehalf-lives for in vivo imaging applications. The single polypeptidechains can be labeled, for example, with radioactive atoms such as ⁹⁰Ty, ^(99M) Tc or ¹¹¹ I via a conjugated metal chelator (see, e.g., Khawet al. ,1980, Science 209: 295; U.S. Pat. No. 4,472,509; U.S. Pat. No.4,479,930), or by other standard means of linking isotopes to proteins,known to those with skill in the art (see for example, Thankur et al.,1991, J. Immunol. Methods 237: 217-224).

The invention is illustrated by the following Examples, which are notintended to be limiting in any way.

EXAMPLES Example 1. Synthesis and Expression of the sFv Constructs(741F8, 26-10 and 520C9).

The construction of several sFv genes using different but standardrecombinant DNA technology, well known to those having ordinary skill inthe art, is described below. These procedures include the amplificationof the V_(H) and V_(L) gene sequences by PCR, the ligation ofappropriate synthetic DNA duplexes and the cloning of V_(H) or V_(L)genes by colony hybridization.

A. 741F8 sFv'.

The V_(H) and V_(L) genes of the 741F8 anti-c-erbB-2 monoclonal antibodywere isolated from the cDNA of the parental 741F8 hybridoma line by PCRusing primers homologous to the N-terminal coding regions of V_(H),V_(L), C_(H) 1, and C_(L). The PCR-amplified V_(H) and V_(L) genes wereisolated by polyacrylamide gel electrophoresis and cloned into a pUCcloning vector. The first FR region of the 741F8 V_(H) gene howevercontained spurious mutations due to the PCR procedure. Errors wererectified by the replacement of the first 70 nucleotides of 741F8 V_(H)with a similar sequence from 520C9 V_(H), another c-erbB-2 specificmonoclonal antibody.

Restriction sites then were introduced into the ends of the heavy andlight chain variable gene segments by site-directed mutagenesis (Kunkelet al., 1985, Proc. Natl. Acad. Sci. USA 82: 488-492). A Nco I siteencoding methionine was positioned at the N-terminus of V_(H) forexpression in E. coli. A Sac I site was created at the 3' end of V_(H)gene. A Xho I site, together with an adjacent Eco RV site, were createdat the N-terminus of V_(L). A stop codon and a Pst I site were placed atthe C-terminal end of V_(L).

The single-chain Fv gene was constructed by connecting the V_(H) andV_(L) genes together with a DNA sequence encoding the 14 residuepolypeptide linker, (Ser₄ Gly)₂ Ser₄, as set forth as amino acids 122through 135 in the Sequence Listing as SEQ. ID. NOS.: 1 and 2.

A synthetic DNA duplex encoding the C-terminal amino acid sequence,(Gly)₄ -Cys was inserted into a Hpa I site located near the stop codonat the 3' end of the 741F8 sFv gene. The resulting 741F8 anti-c-erbB-2sFv' gene was excised from the pUC cloning vector, with the restrictionenzymes Nco I and Bam HI (a Bam HI site is located 3' to the C-terminalPst I site), and inserted into the same sites of a commercial T7expression vector pET-3d (In-vitrogen, Inc.). The resulting gene, setforth in the Sequence Listing as SEQ. ID. NOS.: 1 and 2, was transformedinto E. coli BL21-DE (In-vitrogen, Inc.). Protein expression was inducedby the addition of IPTG to the culture medium.

B. 26-10 sFv'

Construction of the anti-digoxin 26-10 sFv has been described previously(Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85; 5879-5883, and U.S.Pat. No. 5,091,513, both of which are hereby incorporated by reference).Briefly, the synthetic gene was constructed by ligating multiplesynthetic DNA duplexes together. The C-terminal DNA duplex coding forthe amino acid sequence (Gly)₄ -Cys subsequently was ligated into a HpaI restriction site close to the 3' end of the 26-10 sFv gene. Theresulting sFv' gene, set forth in the Sequence Listing as SEQ. ID. NOS.:3 and 4, was then inserted into the E. coli expression vector pET-3d.This plasmid was subsequently transformed into E. coli BL21-DE(In-vitrogen, Inc.) and protein expression induced by the addition ofIPTG to the culture medium.

C. 520C9 sFv.

The 520C9 sFv was generated by linking together the V_(H) and V_(L)genes, cloned from a 520C9 hybridoma cDNA library, with a serine richlinker. Briefly, the V_(H) and V_(L) genes were cloned from the 520C9hybridoma cDNA library using probes directed toward the antibodyconstant (C) and joining (J) regions. Appropriate restriction sites wereintroduced at the ends of each gene by site-directed mutagenesis (Kunkelet al., 1985, Proc. Natl. Acad. Sci. USA 82: 488-492). The V_(H) andV_(L) genes were then ligated together with a serine rich linker. Theresulting 520C9 sFv gene, set forth in the Sequence Listing as SEQ. ID.NOS.: 5 and 6, was transformed into the E. coli expression vector andexpressed as described above and in co-pending U.S. Ser. No. 831,967,incorporated therein by reference.

Example 2. Renaturation, Dimerization and Purification of sFv Proteins.

A. Renaturation and Purification of sFv monomers.

Protocols for renaturing sFv monomers derived from E. coli inclusionbodies are described below. In separate experiments the 7418, 26-10 and520C9 sFv polypeptides were expressed in E. coli. The unfolded sFvproteins were solubilized from inclusion bodies and refolded underappropriate redox conditions. The refolded sFv polypeptide chains werepurified by affinity chromatography or by a combination of ion-exchangeand size exclusion chromatography when affinity chromatography was notfeasible or expedient.

Renaturation of 741F8 sFv'.

Inclusion bodies containing the 741F8 sFv' proteins were washed in abuffer containing 25 mM Tris, 10 mM EDTA, 1.5M GuHCl, pH 8.0 andsolubilized in 25 mM Tris, 10 mM EDTA, 7M GuHCl, pH 9.0 to an OD₂₈₀ nmof about 25-50. The sample was reduced overnight at room temperature bythe addition of dithiothreitol (DTT) to a final concentration of 10 mM.The thiol groups were converted into mixed disulfides with glutathioneby the addition of solid oxidized glutathione to a final concentrationof 100 mM. The solution was adjusted to pH 9.0 and incubated for 4 hr atroom temperature. The 741F8 sFv' polypeptide chains then were refoldedin vitro to generate stable monomers with their C-terminal cysteinesremaining blocked with glutathione. The 741F8 sFv' mixed disulfidepreparation was diluted to an OD₂₈₀ of about 0.15 by the addition of 10mM Tris, 4 mM EDTA, 6M urea, pH 8.5 at 4° C. After two hours an equalvolume of 10 mM Tris, 4 mM EDTA, 1 mM reduced glutathione, pH 8.5,precooled to 4° C., was added with rapid mixing to reduce the ureaconcentration to 3M. After dilution, the samples were allowed torenature for 72 hr at 4° C.

Renaturation of 26-10 sFv'.

Inclusion bodies containing the 26-10 sFv' proteins were washed with 25mM Tris, 10 mM EDTA and solubilized in 6M GuHCl, 25 mM Tris, 10 mM EDTA,pH 8.7 to an OD₂₈₀ nm of about 10 to 20. The dissolved proteins werereduced by overnight incubation at room temperature after the additionof DTT to 10 mM. The reduced protein could also be blocked with oxidizedglutathione as noted above for the 741F8 sFv' polypeptide. The reduced,denatured 26-10 sFv' polypeptides were refolded in a manner similar tothat for the 741F8 sFv' by diluting the preparation into a buffercontaining 3M urea, 0.1 mM oxidized and 0.01 mM reduced glutathione togive a final protein concentration of about 0.15 mg/ml. After overnightincubation at 4° C., the mixture was dialyzed against PBS containing0.05M KH₂ PO₄, 0.15M NaCl, pH 7 for two days at 4° C.

Renaturation of 520C9 sFv.

The inclusion bodies containing the 520C9 sFv were washed with 25 mMTris, 10 mM EDTA, pH 8.0, 1M GuHCl and solubilized in 25 mM Tris, 10 mMEDTA, 6M GuHCl, 10 mM dithiothreitol (DTT), pH 9.0. The material wasethanol precipitated and resuspended in 25 mM Tris, 10 mM EDTA, 6M urea,10 mM DTT, pH 8.0 and fractionated by ion exchange chromatography toremove contaminating nucleic acids and E. coli proteins beforerenaturation of the sFv. The material that did not bind to a DEAESepharose Fast Flow (FF) column was precipitated by lowering the pH to5.5 with 1M acetic acid. The pellet was resolubilized in 25 mM Tris, 10mM EDTA, 6M GuHCl, 10 mM DTT, pH 9.0 and oxidized by overnightincubation at room temperature following dilution into a buffercontaining 25 mM Tris, 10 mM EDTA 6M GuHCl, 1 mM oxidized glutathione,0.1 mM reduced glutathione, pH 9.0. After overnight oxidation the samplewas dialyzed against 10 mM NaH₂ PO₄, 1 mM EDTA, 150 mM NaCl, 500 mMurea, pH 8.0 and the sample clarified by filtration through a membranewith a 100 kD mol. wt. cut-off prior to purification on a c-erbB-2affinity column.

Purification of the refolded sFv Polypeptides.

The refolded 26-10 sFv' polypeptide chains were purified byouabain-Sepharose affinity chromatography, as described for the 26-10sFv constructs (Huston, et. al., 1988, Proc. Natl Acad. Sci. USA 85;5879-5883 and Tai, et al., 1990, Biochem. 29, 8024-3080, both of whichare hereby incorporated by reference). The refolded 520C9 sFvpolypeptide chain was similarly purified using a c-erbB-2-agaroseaffinity column. In this case, the refolded samples were loaded onto ac-erbB-2 affinity column, the column washed with PBS, and the 520C9 sFvpolypeptides eluted with PBS pH 6.1 containing 3M LiCl. The buffer wasthen exchanged by dialysis. The c-erbB-2 affinity column preferably wasprepared by linking the extracellular domain of c-erbB-2 onto agarosebeads.

Briefly, the c-erbB-2 sequence coding for its extracellular domain (ECD)was derived from the baculovirus expression vector described previously(Ring et al., 1992, Mol. Immunol. 28; 915-917). A DNA duplex encodingthe His₆ peptide was ligated to the 3' end of the ECD gene, and theconstruct expressed in CHO cells. The ECD polypeptide was purified fromthe CHO cell culture medium on an IMAC metal affinity column (Pharmacia,Piscataway, N.J.), as described in Skerra, et al., 1991, Bio/Technology9: 273-278, and the eluted ECD proteins attached onto agarose beads togenerate the c-erbB-2-agarose affinity resin.

The renatured 741F8 sFv' polypeptides were purified by a combination ofion exchange and size exclusion chromatography. Briefly, the renatured741F8 sFv' preparation was passed through a DEAE-cellulose column andthe 741F8 sFv' in the unbound fraction adjusted to pH 5.0 before loadingon an S-Sepharose FF column. The 741F8 sFv' polypeptide chains wereeluted with PBS containing 2 mM EDTA and 3M urea, and dialyzed against10 mM Tris, 2 mM EDTA, 20 mM NaCl, pH 7.5 at 20° C. The precipitate washarvested by centrifugation, dissolved in a suitable buffer, and passedthrough a Q-Sepharose FF column. The unbound material was adjusted to pH5.5 and reloaded onto a S-Sepharose FF column. The 741F8 sFv'polypeptides were eluted with a PBS, 2 mM EDTA, 100 mM NaCl, 3M ureabuffer and dialyzed against PBS, 2 mM EDTA. The precipitate washarvested again by centrifugation, dissolved in a suitable buffer,sucrose added to 5% (w/v), and the 741F8 sFv' concentrated to 5 mg/ml ina YM10 membrane concentrator (Amicon). The 741F8 sFv' polypeptide chainswere fractionated by gel filtration chromatography using a S-200 HRcolumn (Pharmacia LKB Biotechnology) and a PBS, 2 mM EDTA buffer.

B. Dimerization of the sFv' constructs

Dimerization of sFv' monomers can be induced using standard crosslinkingconditions. Where disulfide bond formation is desired, the monovalentsFv' polypeptide chains initially are deblocked by mild reduction and(sFv')₂ dimers formed by crosslinking the sFv' polypeptides either bydisulfide linkages or by thioether linkages with the BMH orMCA-peptide-MCA crosslinking reagents.

In order to generate disulfide linked constructs the purified 741F8 and26-10 sFv' preparations were dialyzed against 50 mM Tris, 150 mM NaCl,pH 8.5. The C-terminal glutathionyl blocking groups were removed by theaddition DTT to a concentration of 2 mM followed by overnight incubationat room temperature. Excess reducing agent was removed by extensivedialysis against 50 mM Tris, 150 mM NaCl, pH 8.5, during which themajority of the sFv' polypeptides oxidized into the homodimeric form.

In order to generate BMH and MCA-peptide-MCA crosslinked constructs,sFv' polypeptide chains in PBS first were reduced for two hours at roomtemperature by the addition of DTT to a final concentration of 1 mM. Thesamples were desalted by gel filtration chromatography using a PBS, 1 mMEDTA buffer. A 4-5 fold molar excess of either the BMH orMCA-peptide-MCA linkers, both dissolved in dimethylsulfoxide, were addedto the reduced protein and incubated for at least 12 hours at roomtemperature. The resulting dimers were then purified by HPLC gelfiltration chromatography.

A modification of the procedure of Brennan, et al. (1985, Science 229:81-83) may be used to generate disulfide linked sFv' heterodimers. Forexample, in order to link the 741F8 and 26-10 sFv' polypeptides athionitrobenzoate (TNB) derivative of the 26-10 sFv' (26-10 sFv'-TNB)was mixed with mildly reduced 741F8 sFv'. The 26-10 sFv'-TNB wasprepared by reducing the 26-10 sFv' in PBS with 15 mM2-mercaptoethylamine for 30 minutes at room temperature. The reducingagent was removed by gel filtration and the reduced 26-10 sFv' reactedwith 2.2 mM dithionitrobenzoate (DTNB) for 3 hours. The active 26-10sFv'-TNB was adsorbed onto onto ouabain-Sepharose. The glutathionylblocked 741F8 sFv' monomer in 25 mM Tris, 150 mM NaCl, pH 8.2 wasreduced for 2 hours at room temperature by the addition of DTT to afinal concentration of 1 mM. The excess DTT was removed by gelfiltration and the reduced 741F8 sFv' reacted overnight at roomtemperature with the 26-10 sFv'-TNB complexed to ouabain-Sepharose. Theprogress of the reaction was monitored spectroscopically at 412 nm, theabsorbance maximum of the TNB anion.

C. Purification of (sFv')₂ Dimers.

The (sFv')₂ homodimers may be separated from the sFv' monomers by gelfiltration chromatography. Following dimerization, the sFv' preparationsare dialyzed against PBS containing 1 mM EDTA, 3M urea, 0.03% azide, todisrupt any non-covalent homodimers and fractionated by HPLC on aTSK-G20000SW column using the same buffer. The procedure requires twopasses for purification of the (sFv')₂ homodimers to homogeneity. Thepurified homodimers may be dialyzed either against PBS or any othersuitable buffer prior to use.

The (sFv')₂ heterodimers can be separated by a two step affinitychromatography procedure taking advantage of the bivalent nature of thedimer. For instance, during the the purification of the 741F8/26-10heterodimer the mixture initially was loaded onto an ouabain-Sepharosecolumn, washed with a PBS, 1M NaCl buffer, to remove anynon-specifically adsorbed material, and rewashed with PBS to reduce thesalt concentration. The reactive 26-10 sFv' species bound to the resinwere eluted with 20 mM ouabain in PBS and the eluate dialyzed againstPBS to remove the cardiac glycoside. The 741F8/26-10 heterodimers werethen repurified on a c-erbB-2-agarose affinity column taking advantageof the ECD binding site in the heterodimer. After the preparation wasloaded onto the c-erbB-2 affinity column, it is washed with PBS and the(sFv')₂ heterodimer eluted with 25 mM Tris, 10 mM EDTA, 5M LiCl, pH 6.8.Prior to use, the buffer was exchanged with PBS by dialysis.

Example 3. Immunoreactivity of the Monomeric and Dimeric sFvPolypeptides.

A. Radiolabeling of the sFv' Constructs.

The sFv' polypeptides may be labeled by the chloramine-T method asdescribed (DeNardo, et al., 1986, Nucl. Med. Biol. 13: 303-310).Briefly, 1.0-2.0 mg of sFv' was combined with ¹²⁵ I 14-17 mCi/μg!(Amersham, Arlington Heights, Ill.) at an iodine to protein ratio of1:10 in a 12×75 mm plastic test tube. 10 μl 1 mg/ml! of chloramine-T(Sigma, St. Louis, Mo.) per 100 μg of protein was added and the mixtureincubated for three minutes at room temperature. After the reaction wasterminated, unincorporated ¹²⁵ I was separated from the labeled sFv' bythe spun-column method of Meares, et al., 1984, Anal. Biochem. 142:68-78. Specific activities of 0.2-1.0 mCi/mg for the ¹²⁵ I-labeledproducts may be routinely obtained.

B. Competition ELISA

In order to prepare c-erbB-2, SK-Br-3 breast cancer cells (Ring et al.,1989, Cancer Res. 49: 3070-3080), were harvested and resuspended in 10mM NaCl, 0.5% Nonidet-P40, pH 8. Insoluble debris was removed bycentrifugation and the extract filtered through 0.45 Millex HA and 0.2Millex GV filters. 40 μl of the extract was added to each well of a 96well plate and incubated overnight at 37° C. The plates then were washedwith PBS and non-specific binding sites blocked following the additionof PBS containing 1% skim milk by incubation for one hour at roomtemperature. The sFv and 520C9 Fab samples, diluted in PBS, were addedto the wells and incubated for 30 mins at room temperature. A controlcontaining only dilution buffer was also included.

In order to quantitate the reaction, 20 μl of a 520C9-horseradishperoxidase (HRP) probe (Zymed Labs., South San Francisco, Calif.),diluted to 14 μl/ml in PBS containing 1% skim milk, was added to eachwell and incubated for one hour at room temperature. The plate was thenwashed four times with PBS, the peroxidase substrate added and incubatedfor 30 minutes at room temperature. The reaction was quenched with H₂SO₄ and the OD_(150nm) values measured.

FIG. 3 compares the binding ability of the parental 520C9 Fab fragment,together with the 520C9 sFv single-chain binding protein. The 520C9 sFvsamples included the material obtained following renaturation of thepolypeptide in vitro, a sample purified on a c-erbB-2 agarose affinitycolumn, and the material that did not bind to the column. The fullypurified 520C9 sFv polypeptide exhibits an affinity for c-erbB-2indistinguishable from the parent 520C9 Fab fragment.

C. Biodistribution Studies.

In vivo immunotargeting tissue imaging studies were performed usingstandard procedures. Approximately 2.5×10⁶ SK-OV-3 cells (a humanovarian cancer cell line that expresses c-erbB-2 on the cell surface) inlog phase were implanted subcutaneously onto the hips of four to sixweek old C.B17/ICI-scid mice. Three days after Lugol's solution wasplaced in the drinking water to block the accumulation of radioiodine inthe thyroid, the mice were used in the biodistribution assays.

The radiolabeled sFv' and Fab preparations were diluted in PBS for thesestudies. The biodistribution of the glutathionyl-blocked 741F8 sFv'monomers, and the 741F8 and 26-10 (sFv')₂ constructs were compared afteridentical doses of the radiolabeled protein was administered byinjection in each case. The total injected doses were determined bycounting each animal on a Series 30 multichannel analyzer/probe system(probe model #2007, Canaberra, Meridian, Conn.). Groups of 3-6 mice weresacrificed twenty four hours after injection, the tumors and organs wereremoved, weighed and counted in a gamma counter to determine the amountof radiolabel incorporated into the tissues. From these measurements,the percentage of the initial injected dose incorporated per gram oftissue (%ID/gram) or the amount of label incorporated into the tumorrelative to the amount of radiolabel incorporated into the other organs(T:O ratio) were determined. For specific details see DeNardo, et al.,1977, Cancer, 40: 2923-2929, or Adams, et al., 1992, Antibody,Immunoconjugates, and Radiopharmaceuticals 5: 81-95, both of which arehereby incorporated by reference. Specificity indices also can bedetermined by dividing the T:O ratios of the ¹²⁵ I-741F8 sFv' by thecorresponding T:O ratios of the ¹²⁵ I-26-10 sFv'. The results of thebiodistribution studies 24 hours post administration are summarized inFIGS. 4 and 5. The mean standard error (SEM) for each value is less than30%, except where indicated.

The disulfide linked 741F8 (sFv')₂ homodimers exhibit identical tumorspecificities when compared to the monomeric 741F8 sFv' polypeptidechains. The T:O ratios of the 741F8 sFv' constructs consistently exceedthose for the 26-10 sFv' constructs, demonstrating the bindingspecificity of the 741F8 constructs for the tumors (FIG. 4). Inaddition, the 741F8 (sFv')₂ dimers generally exhibit higher T:O ratiosrelative to that of the monomeric species, particularly for thedisulfide bonded sFv' 741F8 (sFv'-(Gly)₄ Cys)₂ and the MCA linked 741F8(sFv')₂ homodimers. In addition, the 741F8 (sFv')₂ homodimers localizein greater amounts in the tumors relative to the monomeric sFv' species(FIG. 5).

In a separate comparative study with ¹²⁵ I-labeled 26-10 (sFv')₂ and thefollowing species of ¹²⁵ I-labeled 741F8: sFv' monomers, Fab, disulfidelinked (sFv'-Gly₄ Cys)₂ homodimers, and MCA- and BMH-linked (sFv')₂homodimers, the in vivo tumor localization properties of these moleculeswere compared (% ID/gram tumor tissue, see FIG. 6). As is evident fromthe figure, the tumor localization properties of all of the dimeric741F8 (sFv')₂ constructs are significantly greater than those observedwith the 741F8 Fab, the 741F8 sFv' monomer and the 26-10 (sFv')₂ dimer(FIG. 6). The results demonstrate that the increased apparent avidityand enhanced in vivo imaging of the (sFv')₂ dimer is due, at least inpart, to its improved retention in tumor tissue.

EMBODIMENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 909 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..752                                                          (D) OTHER INFORMATION: /product="741F8 sFv'C-terminal                         Gly4-Cys"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCATGGCGGAGATCCAATTGGTGCAGTCTGGACCTGAGCTGAAGAAG47                             MetAlaGluIleGlnLeuValGlnSerGlyProGluLeuLysLys                                 151015                                                                        CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTC95                            ProGlyGluThrValLysIleSerCysLysAlaSerGlyTyrThrPhe                              202530                                                                        ACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTA143                           ThrAsnTyrGlyMetAsnTrpValLysGlnAlaProGlyLysGlyLeu                              354045                                                                        AAGTGGATGGGCTGGATAAACACCAACACTGGAGAGCCAACATATGCT191                           LysTrpMetGlyTrpIleAsnThrAsnThrGlyGluProThrTyrAla                              505560                                                                        GAAGAGTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGC239                           GluGluPheLysGlyArgPheAlaPheSerLeuGluThrSerAlaSer                              657075                                                                        ACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACA287                           ThrAlaTyrLeuGlnIleAsnAsnLeuLysAsnGluAspThrAlaThr                              80859095                                                                      TATTTCTGTGGAAGGCAATTTATTACCTACGGCGGGTTTGCTAACTGG335                           TyrPheCysGlyArgGlnPheIleThrTyrGlyGlyPheAlaAsnTrp                              100105110                                                                     GGCCAAGGGACTCTGGTCACTGTCTCTGCATCGAGCTCCTCCGGATCT383                           GlyGlnGlyThrLeuValThrValSerAlaSerSerSerSerGlySer                              115120125                                                                     TCATCTAGCGGTTCCAGCTCGAGCGATATCGTCATGACCCAGTCTCCT431                           SerSerSerGlySerSerSerSerAspIleValMetThrGlnSerPro                              130135140                                                                     AAATTCATGTCCACGTCAGTGGGAGACAGGGTCAGCATCTCCTGCAAG479                           LysPheMetSerThrSerValGlyAspArgValSerIleSerCysLys                              145150155                                                                     GCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCA527                           AlaSerGlnAspValSerThrAlaValAlaTrpTyrGlnGlnLysPro                              160165170175                                                                  GGGCAATCTCCTAAACTACTGATTTACTGGACATCCACCCGGCACACT575                           GlyGlnSerProLysLeuLeuIleTyrTrpThrSerThrArgHisThr                              180185190                                                                     GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACT623                           GlyValProAspArgPheThrGlySerGlySerGlyThrAspTyrThr                              195200205                                                                     CTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTCATTACTGT671                           LeuThrIleSerSerValGlnAlaGluAspLeuAlaLeuHisTyrCys                              210215220                                                                     CAGCAACATTATAGAGTGCCGTACACGTTCGGAGGGGGGACCAAGCTG719                           GlnGlnHisTyrArgValProTyrThrPheGlyGlyGlyThrLysLeu                              225230235                                                                     GAGATAAAACGGGCTGATGGGGGAGGTGGATGTTAACGGGGGAGGTGGATGTT772                      GluIleLysArgAlaAspGlyGlyGlyGlyCys                                             240245250                                                                     GGGTCTCGTTACGTTGCGGATCTCGAGGCTATCTTTACTAACTCTTACCGTAAAGTTCTG832               GCTCAACTGTCTGCACGCAAGCTTTTGCAGGATATCATGAGCGCTTAAGATCCGTCGACC892               TGCAGGCATGCAAGCTT909                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 250 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaGluIleGlnLeuValGlnSerGlyProGluLeuLysLysPro                              151015                                                                        GlyGluThrValLysIleSerCysLysAlaSerGlyTyrThrPheThr                              202530                                                                        AsnTyrGlyMetAsnTrpValLysGlnAlaProGlyLysGlyLeuLys                              354045                                                                        TrpMetGlyTrpIleAsnThrAsnThrGlyGluProThrTyrAlaGlu                              505560                                                                        GluPheLysGlyArgPheAlaPheSerLeuGluThrSerAlaSerThr                              65707580                                                                      AlaTyrLeuGlnIleAsnAsnLeuLysAsnGluAspThrAlaThrTyr                              859095                                                                        PheCysGlyArgGlnPheIleThrTyrGlyGlyPheAlaAsnTrpGly                              100105110                                                                     GlnGlyThrLeuValThrValSerAlaSerSerSerSerGlySerSer                              115120125                                                                     SerSerGlySerSerSerSerAspIleValMetThrGlnSerProLys                              130135140                                                                     PheMetSerThrSerValGlyAspArgValSerIleSerCysLysAla                              145150155160                                                                  SerGlnAspValSerThrAlaValAlaTrpTyrGlnGlnLysProGly                              165170175                                                                     GlnSerProLysLeuLeuIleTyrTrpThrSerThrArgHisThrGly                              180185190                                                                     ValProAspArgPheThrGlySerGlySerGlyThrAspTyrThrLeu                              195200205                                                                     ThrIleSerSerValGlnAlaGluAspLeuAlaLeuHisTyrCysGln                              210215220                                                                     GlnHisTyrArgValProTyrThrPheGlyGlyGlyThrLysLeuGlu                              225230235240                                                                  IleLysArgAlaAspGlyGlyGlyGlyCys                                                245250                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 779 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..758                                                          (D) OTHER INFORMATION: /product="26-10 sFv'with                               C-terminal Gly4-Cys"                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCATGGAAGTTCAACTGCAACAGTCTGGTCCTGAATTGGTTAAACCT47                             MetGluValGlnLeuGlnGlnSerGlyProGluLeuValLysPro                                 151015                                                                        GGCGCCTCTGTGCGCATGTCCTGCAAATCCTCTGGGTACATTTTCACC95                            GlyAlaSerValArgMetSerCysLysSerSerGlyTyrIlePheThr                              202530                                                                        GACTTCTACATGAATTGGGTTCGCCAGTCTCATGGTAAGTCTCTAGAC143                           AspPheTyrMetAsnTrpValArgGlnSerHisGlyLysSerLeuAsp                              354045                                                                        TACATCGGGTACATTTCCCCATACTCTGGGGTTACCGGCTACAACCAG191                           TyrIleGlyTyrIleSerProTyrSerGlyValThrGlyTyrAsnGln                              505560                                                                        AAGTTTAAAGGTAAGGCGACCCTTACTGTCGACAAATCTTCCTCAACT239                           LysPheLysGlyLysAlaThrLeuThrValAspLysSerSerSerThr                              657075                                                                        GCTTACATGGAGCTGCGTTCTTTGACCTCTGAGGACTCCGCGGTATAC287                           AlaTyrMetGluLeuArgSerLeuThrSerGluAspSerAlaValTyr                              80859095                                                                      TATTGCGCGGGCTCCTCTGGTAACAAATGGGCCATGGATTATTGGGGT335                           TyrCysAlaGlySerSerGlyAsnLysTrpAlaMetAspTyrTrpGly                              100105110                                                                     CATGGTGCTAGCGTTACTGTGAGCTCCTCCGGATCTTCATCTAGCGGT383                           HisGlyAlaSerValThrValSerSerSerGlySerSerSerSerGly                              115120125                                                                     TCCAGCTCGAGTGGATCCGACGTCGTAATGACCCAGACTCCGCTGTCT431                           SerSerSerSerGlySerAspValValMetThrGlnThrProLeuSer                              130135140                                                                     CTGCCGGTTTCTCTGGGTGACCAGGCTTCTATTTCTTGCCGCTCTTCC479                           LeuProValSerLeuGlyAspGlnAlaSerIleSerCysArgSerSer                              145150155                                                                     CAGTCTCTGGTCCATTCTAATGGTAACACTTACCTGAACTGGTACCTG527                           GlnSerLeuValHisSerAsnGlyAsnThrTyrLeuAsnTrpTyrLeu                              160165170175                                                                  CAAAAGGCTGGTCAGTCTCCGAAGCTTCTGATCTACAAAGTCTCTAAC575                           GlnLysAlaGlyGlnSerProLysLeuLeuIleTyrLysValSerAsn                              180185190                                                                     CGCTTCTCTGGTGTCCCGGATCGTTTCTCTGGTTCTGGTTCTGGTACT623                           ArgPheSerGlyValProAspArgPheSerGlySerGlySerGlyThr                              195200205                                                                     GACTTCACCCTGAAGATCTCTCGTGTCGAGGCCGAAGACCTGGGTATC671                           AspPheThrLeuLysIleSerArgValGluAlaGluAspLeuGlyIle                              210215220                                                                     TACTTCTGCTCTCAGACTACTCATGTACCGCCGACTTTTGGTGGTGGC719                           TyrPheCysSerGlnThrThrHisValProProThrPheGlyGlyGly                              225230235                                                                     ACCAAGCTCGAGATTAAACGTTCCGGGGGAGGTGGATGTTAACTGCAGC768                          ThrLysLeuGluIleLysArgSerGlyGlyGlyGlyCys                                       240245250                                                                     CCGGGGGATCC779                                                                (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 252 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGluValGlnLeuGlnGlnSerGlyProGluLeuValLysProGly                              151015                                                                        AlaSerValArgMetSerCysLysSerSerGlyTyrIlePheThrAsp                              202530                                                                        PheTyrMetAsnTrpValArgGlnSerHisGlyLysSerLeuAspTyr                              354045                                                                        IleGlyTyrIleSerProTyrSerGlyValThrGlyTyrAsnGlnLys                              505560                                                                        PheLysGlyLysAlaThrLeuThrValAspLysSerSerSerThrAla                              65707580                                                                      TyrMetGluLeuArgSerLeuThrSerGluAspSerAlaValTyrTyr                              859095                                                                        CysAlaGlySerSerGlyAsnLysTrpAlaMetAspTyrTrpGlyHis                              100105110                                                                     GlyAlaSerValThrValSerSerSerGlySerSerSerSerGlySer                              115120125                                                                     SerSerSerGlySerAspValValMetThrGlnThrProLeuSerLeu                              130135140                                                                     ProValSerLeuGlyAspGlnAlaSerIleSerCysArgSerSerGln                              145150155160                                                                  SerLeuValHisSerAsnGlyAsnThrTyrLeuAsnTrpTyrLeuGln                              165170175                                                                     LysAlaGlyGlnSerProLysLeuLeuIleTyrLysValSerAsnArg                              180185190                                                                     PheSerGlyValProAspArgPheSerGlySerGlySerGlyThrAsp                              195200205                                                                     PheThrLeuLysIleSerArgValGluAlaGluAspLeuGlyIleTyr                              210215220                                                                     PheCysSerGlnThrThrHisValProProThrPheGlyGlyGlyThr                              225230235240                                                                  LysLeuGluIleLysArgSerGlyGlyGlyGlyCys                                          245250                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 739 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..729                                                          (D) OTHER INFORMATION: /product="520C9 sFv polypeptide                        sequence"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAGATCCAATTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAG48                            GluIleGlnLeuValGlnSerGlyProGluLeuLysLysProGlyGlu                              151015                                                                        ACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCGCAAACTAT96                            ThrValLysIleSerCysLysAlaSerGlyTyrThrPheAlaAsnTyr                              202530                                                                        GGAATGAACTGGATGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATG144                           GlyMetAsnTrpMetLysGlnAlaProGlyLysGlyLeuLysTrpMet                              354045                                                                        GGCTGGATAAACACCTACACTGGACAGTCAACATATGCTGATGACTTC192                           GlyTrpIleAsnThrTyrThrGlyGlnSerThrTyrAlaAspAspPhe                              505560                                                                        AAGGAACGGTTTGCCTTCTCTTTGGAAACCTCTGCCACCACTGCCCAT240                           LysGluArgPheAlaPheSerLeuGluThrSerAlaThrThrAlaHis                              65707580                                                                      TTGCAGATCAACAACCTCAGAAATGAGGACTCGGCCACATATTTCTGT288                           LeuGlnIleAsnAsnLeuArgAsnGluAspSerAlaThrTyrPheCys                              859095                                                                        GCAAGACGATTTGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCAGT336                           AlaArgArgPheGlyPheAlaTyrTrpGlyGlnGlyThrLeuValSer                              100105110                                                                     GTCTCTGCATCGATATCGAGCTCCTCCGGATCTTCATCTAGCGGTTCC384                           ValSerAlaSerIleSerSerSerSerGlySerSerSerSerGlySer                              115120125                                                                     AGCTCGAGTGGATCCGATATCCAGATGACCCAGTCTCCATCCTCCTTA432                           SerSerSerGlySerAspIleGlnMetThrGlnSerProSerSerLeu                              130135140                                                                     TCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAG480                           SerAlaSerLeuGlyGluArgValSerLeuThrCysArgAlaSerGln                              145150155160                                                                  GACATTGGTAATAGCTTAACCTGGCTTCAGCAGGAACCAGATGGAACT528                           AspIleGlyAsnSerLeuThrTrpLeuGlnGlnGluProAspGlyThr                              165170175                                                                     ATTAAACGCCTGATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCC576                           IleLysArgLeuIleTyrAlaThrSerSerLeuAspSerGlyValPro                              180185190                                                                     AAAAGGTTCAGTGGCAGTCGGTCTGGGTCAGATTATTCTCTCACCATC624                           LysArgPheSerGlySerArgSerGlySerAspTyrSerLeuThrIle                              195200205                                                                     AGTAGCCTTGAGTCTGAAGATTTTGTAGTCTATTACTGTCTACAATAT672                           SerSerLeuGluSerGluAspPheValValTyrTyrCysLeuGlnTyr                              210215220                                                                     GCTATTTTTCCGTACACGTTCGGAGGGGGGACCAACCTGGAAATAAAA720                           AlaIlePheProTyrThrPheGlyGlyGlyThrAsnLeuGluIleLys                              225230235240                                                                  CGGGCTGATTAATCTGCAG739                                                        ArgAlaAsp                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 243 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GluIleGlnLeuValGlnSerGlyProGluLeuLysLysProGlyGlu                              151015                                                                        ThrValLysIleSerCysLysAlaSerGlyTyrThrPheAlaAsnTyr                              202530                                                                        GlyMetAsnTrpMetLysGlnAlaProGlyLysGlyLeuLysTrpMet                              354045                                                                        GlyTrpIleAsnThrTyrThrGlyGlnSerThrTyrAlaAspAspPhe                              505560                                                                        LysGluArgPheAlaPheSerLeuGluThrSerAlaThrThrAlaHis                              65707580                                                                      LeuGlnIleAsnAsnLeuArgAsnGluAspSerAlaThrTyrPheCys                              859095                                                                        AlaArgArgPheGlyPheAlaTyrTrpGlyGlnGlyThrLeuValSer                              100105110                                                                     ValSerAlaSerIleSerSerSerSerGlySerSerSerSerGlySer                              115120125                                                                     SerSerSerGlySerAspIleGlnMetThrGlnSerProSerSerLeu                              130135140                                                                     SerAlaSerLeuGlyGluArgValSerLeuThrCysArgAlaSerGln                              145150155160                                                                  AspIleGlyAsnSerLeuThrTrpLeuGlnGlnGluProAspGlyThr                              165170175                                                                     IleLysArgLeuIleTyrAlaThrSerSerLeuAspSerGlyValPro                              180185190                                                                     LysArgPheSerGlySerArgSerGlySerAspTyrSerLeuThrIle                              195200205                                                                     SerSerLeuGluSerGluAspPheValValTyrTyrCysLeuGlnTyr                              210215220                                                                     AlaIlePheProTyrThrPheGlyGlyGlyThrAsnLeuGluIleLys                              225230235240                                                                  ArgAlaAsp                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "Linker 1"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "LINKER 2"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       SerSerSerSerGlySerSerSerSerGlySerSerSerSerGly                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "C-Terminal Tail (Ser-Cys)"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerCys                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "C-Terminal Tail                                (Gly-Gly-Gly-Gly-Cys)"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyGlyGlyGlyCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: /note= "C-Terminal Tail                                (His-His-His-His-His-His-Gly-Gly-Gly-Gly-Cys)"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisHisHisHisHisHisGlyGlyGlyGlyCys                                             1510                                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid sequence encoding apolypeptide chain that binds preferentially to a preselected antigen,the polypeptide chain comprising:an amino acid sequence defining,(a) ansFv comprising two polypeptide domains connected by a polypeptide linkerspanning the distance between the C-terminus of one domain and theN-terminus of the other, the amino acid sequence of each said domaincomprising complementarity determining regions (CDRs) interposed betweenframework regions (FRs), the CDRs and FRs of said sFv together defininga binding site that binds preferentially to said preselected antigen,and (b) a C-terminal tail peptide bonded to the C-terminus of the sFvand having an amino acid sequence selected from the group consisting ofSer-Cys, (Gly)₄ -Cys, and (His)₆ -(Gly)₄ -Cys.
 2. The isolated nucleicacid of claim 1, wherein the C-terminal tail comprises the amino acidsequence Ser-Cys.
 3. The isolated nucleic acid of claim 1, wherein theC-terminal tail comprises the amino acid sequence (Gly)₄ -Cys.
 4. Theisolated nucleic acid of claim 1, wherein the C-terminal tail comprisesthe amino acid sequence (His)₆ -(Gly)₄ -Cys.
 5. The isolated nucleicacid of claim 1, wherein the CDR sequences are derived from animmunoglobulin that binds c-erbB-2 or a c-erbB-2-related antigen.
 6. Theisolated nucleic acid of claim 5, wherein the CDR sequences are derivedfrom an immunoglobulin selected from the group consisting of themonoclonal antibodies 520C9, 741F8, and 454C11.
 7. The isolated nucleicacid of claim 1 defined by the DNA sequence set forth in Sequence IDNo.
 1. 8. A host cell transfected with the isolated nucleic acid ofclaim
 1. 9. A host cell transfected with the isolated nucleic acid ofclaim
 5. 10. A host cell transfected with the isolated nucleic acid ofclaim
 2. 11. A host cell transfected with the isolated nucleic acid ofclaim
 3. 12. A host cell transfected with the isolated nucleic acid ofclaim 4.